The author thanks Susan Whitfield for preparation of the figures and Camille Martin and Hillary Samagaio for assistance with some of the experiments.

<ol>
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The author declares that she has no competing financial interests.

It is important to be aware of all of the surfaces to which bacteria can adhere during the experiment. Thus bacteria which are capable of binding to glass may be underestimated if viable cell counts are done using glass tubes and pipettes. If plants are grown in agar or soil and some of the agar or soil remains on the plants then the bacteria may bind to the adhering material rather than to the plants. On the other hand, washing the plant surface, particularly in the case of roots, may remove natural surface coatings such as mucous and thus alter the results of adherence tests.
It is important to be certain that the bacteria added to the incubation mixture remain alive during the experiment. Thus viable cell counts of free as well as attached bacteria should routinely be made. Some treatments or bacterial mutations may reduce bacterial growth rate or actually cause the death of a fraction of the bacterial population. Live and dead bacteria may not be distinguishable in the microscope unless special stains are used. There is a useful stain kit for live/dead bacteria which depends on the exclusion of dyes from the living bacteria. However, if a mixed population of bacterial species is present then viable cell counts of the species of interest is likely to be the easiest method to determine whether the incubation has resulted in bacterial death.
Medium composition will influence bacterial survival and growth. Root exudate and materials released from wound and cut sites will provide substrate to support modest bacterial growth. Phosphate, nitrogen, and iron tend to be limiting in these conditions. Divalent cations such as calcium and magnesium may influence adhesion. In some cases carbon source can influence adhesion. pH also matters. In general the pH of the rhizosphere is between 5.5 and 6.5.
It is necessary to be careful in using bacteria marked with antibiotic resistance. The antibiotics most often used are rifampicin and nalidixic acid. Resistance to these antibiotics is generally due to mutations in chromosomal genes (RNA polymerase and gyrase, respectively) and thus cannot easily be transferred to another strain during the incubation. This type of resistance also does not result in the degradation or modification of the antibiotic. Marking bacteria with a plasmid-borne gene marker is not recommended unless the plasmid cannot be transferred to any other bacteria. The antibiotic resistance must not be due to degradation or chemical modification of the antibiotic as antibiotic sensitive bacteria will then be able to grow on antibiotic plates if they are located close to the resistant bacteria.
The methods described in this paper are useful for small sample sizes and/or experiments where the samples need to be contained (for example, experiments involving human pathogens). For large sample sizes (above 100 g of material or more than 50 plants) other methods or drastic modification of these methods would be needed10,19,32,19,33,34,35,36. The presence of large numbers of microorganisms other than the species being studied can also pose significant problems. Possible solutions include the use of bacteria marked with a fluorescent protein or antibiotic resistance as described in steps 6.1.2 and 7.3. However, when the bacteria of interest are rare individuals in a large population of other microorganisms these markers may not be adequate to allow an unambiguous assessment of the numbers of the bacteria being studied.
All of the methods described here are laboratory based methods. Minor modifications would be needed for greenhouse studies. More major modifications are likely to be required for field studies where protozoa, insect and other animal predation and climate variation complicate the provision of defined conditions for the experiments. In the future these methods may be extended to include the interactions of two or more microorganisms on the plant surface.

The measurement of bacterial binding to plant surfaces has become important in three disparate situations. The first situation is the examination of the transmission of human pathogens on plant surfaces1,2,3. The goal here is to prevent bacterial binding or to remove or kill bound bacteria and thus to reduce the transmission of disease by plant material. The second situation is the examination of the binding of plant pathogens to plant surfaces4. Once again the goal here is to prevent binding or to remove or kill bound bacteria and thus to reduce disease. The third situation is the examination of the binding of symbiotic or plant-growth-promoting bacteria5,6. The goal here is to promote bacterial binding and thus to increase plant health and crop yields.
The techniques for measuring bacterial binding to plant surfaces described in this article are inexpensive and relatively easy to carry out. The only requirements are a microscope and materials generally found in a bacteriology laboratory. For some techniques a bath sonicator is useful. The techniques described are designed for binding experiments carried out using relatively small sample sizes. Measurements of binding are made in the laboratory, although it may be possible to modify some of these techniques for use in the greenhouse or in the field.
These techniques have been used to measure bacterial binding to roots, sprouts, cut leaves, cut fruits, and intact cherry tomatoes in the laboratory7,8,9,10,11,12,13,14,15. They have also been used to measure root colonization of plants growing in soil or sand in the laboratory16. The techniques have been used with many bacterial species including Agrobacterium tumefaciens, Sinorhizobium meliloti, Escherichia coli, Salmonella enterica, and Pseudomonas fluorescens. A useful description of methods for assessing the interaction of A. tumefaciens with surfaces can be found in Morton and Fuqua (2012)17. In all cases the sample sizes involved were small, generally less than 25 - 50 plants. The techniques described are suitable for use with human pathogens which need to be kept contained during the experiments.

<table><tbody><tr><td>light microscope</td><td>any</td><td>N/A</td><td>phase contrast or Nomarski optics are helpful, for studies using fluorescent markers a fluorescence microscope is required.</td></tr><tr><td>seeds</td><td>any&amp;nbsp;</td><td>N/A</td><td>make a note of the seed lot number and the cultivar</td></tr><tr><td>bleach</td><td>any</td><td>N/A</td><td></td></tr><tr><td>bath sonicator</td><td>any scientific supply company</td><td>N/A</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>nutrient agar&amp;nbsp;</td><td>Difco&amp;nbsp;</td><td>001-01-8</td><td></td></tr><tr><td>soytone</td><td>Difco&amp;nbsp;</td><td>24360</td><td></td></tr><tr><td>Sand</td><td>sea sand Fisher Scientific</td><td>S25</td><td></td></tr><tr><td>sand</td><td>&amp;nbsp;Sigma-Aldrich</td><td>S9887</td><td></td></tr><tr><td>conetainers</td><td>Stuewe &amp;amp; Sons, Inc.</td><td>Ray-Leach cone-tainers</td><td>many different sizes are available to suit the type of plant you wish to grow</td></tr><tr><td>parafilm</td><td>any scientific supply company</td><td>N/A</td><td></td></tr><tr><td>MS salts</td><td>Sigma-Aldrich</td><td>M5524</td><td></td></tr><tr><td>parrafin</td><td>any</td><td>N/A</td><td></td></tr><tr><td>centrigfuge</td><td>any scientific company</td><td>N/A</td><td>to pellet most bacteri only a small centrifuge with a max force of 10,000 XG is needed</td></tr><tr><td>vortex mixer</td><td>any scientific company</td><td></td><td></td></tr><tr><td>micrometer</td><td>ACCU-SCOPE Accessories</td><td>A3145</td><td></td></tr><tr><td>Sedgwick-rafter counting cell</td><td>Hauser Scientific</td><td>HS3800</td><td></td></tr><tr><td>probe-clip press-seal incubatin chamber</td><td>Sigma-Aldrich</td><td>Z359483</td><td></td></tr><tr><td>rifampicin</td><td>Sigma-Aldrich</td><td>R3501</td><td></td></tr><tr><td>naladixic acid</td><td>Sigma-Aldrich</td><td>n8878</td><td></td></tr><tr><td>Live/dead stain</td><td>In Vitrogen Molecular Bioprobes</td><td>L7007</td><td></td></tr></tbody></table>

adherence of bacteria to plant surfaces measured in the laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

A. tumefaciens colonizes root surfaces. In order to determine whether bacterial production of cellulose plays a role in root colonization, the effects of bacterial mutations which prevent cellulose synthesis were examined16. The techniques described in steps 1.3 and 7.1 were used. Tomato seeds were surface sterilized and germinated in sterile water. When the roots were approximately 2 cm long they were dipped in a suspension of 105 bacteria per mL and planted in pasteurized soil in containers. The plants were grown for 14 days at 25 &#176;C on a 12 h light/12 h dark cycle. After the indicated times the plants were removed from the containers. The roots were washed and sonicated in a bath sonicator to remove bound bacteria. Bacterial numbers were determined using viable cell counts. Figure 2 shows the effect of two different cellulose-minus mutations on the ability of the bacteria to colonize tomato roots. Although the standard deviations of some measurements were as high as 0.9 log10 (a common problem with this type of measurement) the reduction in binding of the cellulose-minus mutants is clearly evident and we can conclude that bacterial production of cellulose aids the bacteria in the colonization of tomato roots.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56599/56599fig2.jpg" /> 
Figure 2: Root colonization by wild type and cellulose-minus mutants of&#160;A. tumefaciens.&#160;Log10&#160;total number of bacteria per cm root length recovered from tomato roots inoculated with wild type&#160;A. tumefaciens&#160;strain C58 and cellulose-minus mutants C58:1 and C58:A60. The numbers shown are the means from a minimum of four separate experiments. Bars indicate standard deviations of the means. Roots were inoculated by dipping them in a suspension of 105&#160;bacteria per mL for one min. The plants were grown in containers and the loosely adherent bacteria were removed by washing the roots in washing buffer in a glass vial. Tightly adherent bacteria were removed using a bath sonicator and the resulting suspension plated to determine viable cell counts16. This figure has been modified from Matthysse and McMahan.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56599/56599fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The role of exopolysaccharides in the binding of E. coli and other bacteria to alfalfa sprouts was examined. Some outbreaks of diarrheal disease due to E. coli O157:H7 have been traced to contaminated alfalfa sprouts. Binding of the wild-type bacteria and mutants unable to make various exopolysaccharides was measured using the methods described in steps 1.1, 5.1, and 7.2. Alfalfa sprouts were surface sterilized and germinated for one day in sterile water at 25 &#176;C in the dark. Four sprouts with attached seed coats were placed in sterile plastic dishes containing 5 mL of water. Bacteria grown in Luria broth were added to a final concentration of approximately 5 x 103 per mL. The inoculated sprouts were incubated at 25 &#176;C in the dark for 3 days. The sprouts were washed twice in 5 mL sterile water in a vial by vigorous inversion and homogenized in washing buffer using a motor-driven Teflon glass homogenizer. Previous experiments using bacteria marked with a plasmid carrying the GFP gene showed no internalized bacteria although surface bacteria were easily observed. The results are shown in Table 1. Two strains of E. coli O157:H7 were examined. In both strains the production of poly-&#946;-1,6-glucuronic acid(PGA) appeared to make the largest contribution to the binding of pathogenic E. coli to plant surfaces. Colonic acid also played a significant role in binding. While the reduction in binding in cellulose-minus mutants was significant it was not as large as that for the other two polysaccharides.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td colspan="4">Effects of Mutations in Exopolysaccharide Production Genes on Binding of E. coli O157:H7 to sprouts and open seed coats</td>
		</tr>
		<tr>
			<td rowspan="2">Bacterial strain&#160;</td>
			<td rowspan="2">Mutation or genotype (relevant phenotype)</td>
			<td colspan="2">Log10 &#160;number of bacteria bound per sprout or seed coat&#160;</td>
		</tr>
		<tr>
			<td>Alfalfa sproutsb</td>
			<td>Open seed coats</td>
		</tr>
		<tr>
			<td>86-24</td>
			<td>None (wild type)</td>
			<td>4.7 &#177; 0.6</td>
			<td>5.6 &#177;&#160; 0.2</td>
		</tr>
		<tr>
			<td>8624N</td>
			<td>yhjN (cellulose-minus)</td>
			<td>2.9&#160; &#177;&#160; 0.7c</td>
			<td>3.5&#160; &#177;&#160; 0.6c</td>
		</tr>
		<tr>
			<td>8624C</td>
			<td>wcaD (colonic acid-minus)</td>
			<td>1.8&#160; &#177;&#160; 0.7c</td>
			<td>2.4&#160; &#177;&#160; 0.5c</td>
		</tr>
		<tr>
			<td>8624P</td>
			<td>pgaC (PGA-minus)</td>
			<td>&#60;1.0c</td>
			<td>1.0&#160; &#177;&#160; 1.0c</td>
		</tr>
		<tr>
			<td>DEC4A</td>
			<td>None (wild type)</td>
			<td>5.6&#160; &#177; 0.2</td>
			<td>6.1&#160;&#160; &#177;&#160; 0.3</td>
		</tr>
		<tr>
			<td>DEC4AN</td>
			<td>yhjN (cellulose-minus)</td>
			<td>4.8&#160;&#160; &#177; 0.8d</td>
			<td>4.1&#160;&#160; &#177; 0.8d</td>
		</tr>
		<tr>
			<td>DEC4AC</td>
			<td>wcaD (colonic acid-minus)</td>
			<td>3.9&#160;&#160; &#177; 0.5c</td>
			<td>4.8&#160;&#160; &#177; 0.8d</td>
		</tr>
		<tr>
			<td>DEC4AP</td>
			<td>pgaC (PGA-minus)</td>
			<td>&#60;1.0c</td>
			<td>1.2&#160;&#160; &#177; 0.7c</td>
		</tr>
		<tr>
			<td colspan="4">a mean &#177; standard deviation of a minimum of three measurements of the number (log10) of bacteria bound after 3 days.</td>
		</tr>
		<tr>
			<td colspan="4">b Sprouts were washed before measurement.</td>
		</tr>
		<tr>
			<td colspan="4">c Significantly different from the wild type: P &#60;0.01.</td>
		</tr>
		<tr>
			<td colspan="4">d&#160; significantly different from the wild type: P &#60;0.05.</td>
		</tr>
		<tr>
			<td colspan="4">This table has been modified from&#160; Matthysse, Deora, Mishra, and Torres10.&#160;&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Effects of mutations in exopolysaccharide production genes on binding of&#160;E. coli&#160;O157:H7 to sprouts. In order to determine the role of various exopolysaccharides and lipopolysaccharide in the binding of pathogenic&#160;E. coli&#160;O157:H7 strains to alfalfa sprouts, the binding of a set of mutants to the sprouts and open seed coats was examined using the methods described in step 6. The results showed that poly-&#223;-1, 6-N-acetyl-D-glucosamine (PGA) appears to be essential for binding to sprouts and that both cellulose and colanic acid are required for maximum binding of&#160;E. coli&#160;O157. This table has been modified from Matthysse, Deora, Mishra, and Torres10.
In order to determine if the production of PGA is sufficient to cause bacterial binding to plant surfaces, a plasmid (pMM11) carrying the cloned operon encoding the genes required for PGA production was introduced into two different bacteria which would not ordinarily be able to bind to tomato roots10. A. tumefaciens A1045 is a mutant of the wild type strain C58 which fails to make cyclic-&#946;-1,2 glucan and also fails to bind to plant surfaces29. Sinorhizobium meliloti 1021 which forms root nodules on alfalfa fails to bind to non-legumes including tomato12. The techniques described in steps 1.1, 5.1, 6.3, and 7.1 were used to determine if the ability to make PGA generally increased bacterial binding to root surfaces. Tomato seeds were surface sterilized and germinated in sterile water. Roots were cut into segments 1 cm in length and placed in sterile water and the bacteria were inoculated. As these two species of bacteria grow at different rates, binding was measured at different times to allow for roughly equal amounts of bacterial growth. The presence of the plasmid pMM11 caused a similar significant increase in the number of bound bacteria of both species (Table 2)10. A significant increase in binding was also seen in the light microscope but the binding was very different for the two species (Figure 3). A. tumefaciens A1045 bound as individual bacteria to the root surface. S. meliloti bound in large clusters in which only a few of the bacteria were directly attached to the root and the majority of the bacteria were attached to other bacteria. This example shows that simply analyzing the numbers of bacteria bound without including microscopic observations can give a misleading impression of the results of an experiment. Although both methods (viable cell counts and microscopic observations) show that pMM11 increased bacterial binding to tomato roots, the type of binding caused by the production of PGA was different for the two bacterial species10.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td colspan="3">The Effect of the Plasmid pMM11 on the Binding of Bacteria to Tomato Roots</td>
		</tr>
		<tr>
			<td>Bacterial strain</td>
			<td>Plasmid</td>
			<td>Number of bacteria bound per mm root length</td>
		</tr>
		<tr>
			<td>A. tumefaciens A1045a</td>
			<td>none</td>
			<td>0.25 x 103 &#177; 0.25 x 103</td>
		</tr>
		<tr>
			<td></td>
			<td>pBBR1mcs (vector)</td>
			<td>0.25 x 103 &#177; 0.25 x 103</td>
		</tr>
		<tr>
			<td></td>
			<td>pMM11 (PGA synthesis)</td>
			<td>10 x 103 &#177; 0.25 x 103</td>
		</tr>
		<tr>
			<td>S. meliloti 1021b</td>
			<td>none</td>
			<td>none detected</td>
		</tr>
		<tr>
			<td></td>
			<td>pBBR1mcs (vector)</td>
			<td>none detected</td>
		</tr>
		<tr>
			<td></td>
			<td>pMM11 (PGA synthesis)</td>
			<td>50 x 103 &#177; 5 x 103</td>
		</tr>
		<tr>
			<td colspan="3">a Bacterial binding was measured after 2 hours</td>
		</tr>
		<tr>
			<td colspan="3">b Bacterial binding was measure after 18 hours&#160;</td>
		</tr>
		<tr>
			<td colspan="3">This table has been modified from&#160; Matthysse, Deora, Mishra, and Torres10.&#160;&#160;</td>
		</tr>
	</tbody>
</table>
Table 2: The effect of a plasmid carrying genes for the synthesis of PGA on binding of&#160;A. tumefaciens&#160;A1045 and&#160;S. meliloti&#160;1021 to tomato root segments. In order to examine the ability of poly-&#223;-1, 6-N-acetyl-D-glucosamine (PGA) to promote the binding of bacteria to plant roots, the effect of a plasmid conferring the ability to make PGA (pMM11) on the binding of two strains of plant-associated bacteria to tomato roots was examined. Neither strain of bacteria showed significant binding to tomato roots in the absence of the plasmid or in the presence of the plasmid without the genes encoding PGA synthesis (pBBR1mcs). The addition of the plasmid carrying PGA synthesis genes increased binding by both types of bacteria. Because&#160;A. tumefaciens&#160;grows faster than&#160;S. meliloti&#160;binding was measured after 2 h of incubation for&#160;A. tumefaciens&#160;and after 18 h for&#160;S. meliloti. The techniques used are those described in step 7. This table has been modified from Matthysse, Deora, Mishra, and Torres10.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56599/56599fig3.jpg" /> 
Figure 3: The effect of the plasmid pMM11 carrying the PGA biosynthesis genes on the binding of&#160;A. tumefaciens&#160;A1045 and&#160;S. meliloti&#160;1021 to tomato root hairs.&#160;Binding to tomato root hairs of A)&#160;A. tumefaciens&#160;A1045, B)&#160;A. tumefaciens&#160;A1045 pMM11, C)&#160;S. meliloti&#160;1021, and D)&#160;S. meliloti&#160;1021 pMM11. Although the increase in binding of the two bacterial species is roughly similar the details of the binding as seen in the light microscope are quite different. The techniques used are those described in step 6. This figure has been modified from Matthysse, Deora, Mishra, and Torres10.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56599/56599fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
It is sometimes possible to use binding to non-biological surfaces to aid in distinguishing the contribution of the bacteria and of the plant in a specific interaction. The unipolar polysaccharide(UPP) of A. tumefaciens has been shown to be able to mediate bacterial binding to a variety of both biological and non-biological surfaces30. Calcium was observed to inhibit the binding of A. tumefaciens to plant surfaces mediated by the UPP28. In order to determine whether the inhibition by calcium ions of bacterial binding to plant surfaces is due to an effect on the bacteria or on the plant surface, the binding of the bacteria to nylon threads was examined. The techniques describes in step 8.2 were used. Tomato seeds were surface sterilized and germinated in water as described in step 1. The bacteria were grown in minimal medium with sucrose and added to tomato roots or nylon threads at a final concentration of approximately 105/mL as described in step 5.1. The effect of added CaCl2 on bacterial binding to tomato roots and nylon threads was examined in the microscope. Figure 4 shows a similar inhibition of binding by calcium using either surface suggesting that the effect of calcium is primarily on the bacteria10.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56599/56599fig4.jpg" /> 
Figure 4: The effect of calcium on the binding of A. tumefaciens to tomato root hairs and nylon threads. A. tumefaciens was incubated with tomato roots (A and B) or nylon threads (C and D) for 24 h in a 1:10 dilution of MS salts and a 1:20 dilution of AB minimal medium, 0.4 % sucrose (A and C) or in a 1:10 dilution of MS salts and 1:20 dilution of AB minimal medium, 0.4 % sucrose containing 60 mM CaCl2 (B and D)31. The added CaCl2 inhibited bacterial binding to both roots and nylon threads suggesting that the inhibition was primarily due to an effect on the bacteria rather than on the plant surface. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56599/56599fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Adherence of Bacteria to Plant Surfaces Measured in the Laboratory at JoVE.com

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

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Nanyang Technological University

Research

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12.6K Views.  University of North Carolina at Chapel Hill. Een eenvoudige methode voor het meten en karakteriseren van bacteriële hechting op planten, met name wortels en spruiten, wordt in dit artikel beschreven.

Video: Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Immunology and Infection

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Bioengineering

Rhizobacterial Colonization Detection on Arabidopsis thaliana

Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized method for measuring bacterial colonization in the rhizosphere is necessary to improve reproducibility. We first cultured sterile A.thaliana in hydroponic conditions and then inoculated the bacterial cells in the rhizosphere at a final concentration of OD600 of 0.01. At 2 days post-inoculation, the root tissue was harvested and washed three times in sterile water to remove the uncolonized bacterial cells. The roots were then weighed, and the bacterial cells colonized on the root were collected by vortex. The cell suspension was diluted in a gradient with a phosphate-buffered saline (PBS) buffer, followed by plating onto a Luria-Bertani (LB) agar medium. The plates were incubated at 37 &#176;C for 10 h, and then, the single colonies on LB plates were counted and normalized to indicate the bacterial cells colonized on roots. This method is used to detect bacterial colonization in the rhizosphere in mono-interaction conditions, with good reproducibility.

Author Spotlight: Enhancing Rhizobacteria Colonization on Plant Roots for Improved Microbial Fertilizer Efficiency

Colonization of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere is essential for its growth-promoting effect. It is necessary to standardize the method of detection of bacterial rhizosphere colonization. Here, we describe a reproducible method for quantifying bacterial colonization on the root surface.

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

Use of Fimbrial Rod for F18ab Fimbriae+ STEC Colonization to Host Cells

Type 1 fimbriae are important virulence determinants of some Gram-negative pathogens, which promote bacterial colonization. The fimbrial rod is primarily composed of multiple copies of the major fimbrial subunit FimA. FimH adhesin, however, is present as a fibrillar tip structure that drive bacteria binding to host cellular mannose containing receptor. Here, we provide protocols to evaluate and compare the function of type 1 fimbrial subunits in F18ab fimbriae+ Shiga toxin-producing Escherichia coli (STEC). We found that both FimA and FimH are required for bacterial adhesion, invasion, and biofilm formation. Deleting fimA gene showed much more reduction in bacterial adhesion and invasion to porcine intestinal columnar epithelial cells IPEC-J2, than that of fimH mutant. Biofilm formation was significantly reduced in both mutants with an equal level. In addition, qPCR demonstrated that either fimA or fimH deletion down-regulated the bacterial flagella and F18 fimbriae genes expression, while up-regulated adhesin was involved in diffuse adherence-I (AIDA-I) gene expression, suggesting the co-regulation of cell surface-localized adhesins in F18ab fimbriae+ STEC.

Use of Fimbrial Rod for F18ab Fimbriae+ STEC Colonization to Host Cells

Here we present a protocol to study the function of fimbriae in bacterial colonization.

Type 1 fimbriae are important virulence determinants of some Gram-negative pathogens, which promote bacterial colonization. The fimbrial rod is primarily ...

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly developing resistance to the first and the second line of antifungals. Cp is frequently isolated from hands and skin surfaces, as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to the body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and allow for the fluorescent quantification of individual isolates. We also demonstrate that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

Adhesion is an important first step in colonization and pathogenesis for Candida. Here, an in vitro assay is described to measure adhesion of C. parapsilosis isolates to immobilized proteins under fluid shear. A multichannel microfluidics device is used to compare multiple samples in parallel, followed by quantification using fluorescence imaging.

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly ...

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in ...

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

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